Use of erythropoietin

ABSTRACT

The present invention relates to the use of erythropoietin for stimulating the physiological mobilization, proliferation and differentiation of endothelial progenitor cells, for stimulating vasculogenesis, for the therapy of diseases associated with a dysfunction of endothelial progenitor cells and for producing pharmaceutical compositions for the treatment of such diseases, and pharmaceutical compositions which comprise erythropoietin and other suitable active ingredients for stimulating endothelial progenitor cells.

The present invention relates to the use of erythropoietin forstimulating the physiological mobilization, proliferation anddifferentiation of endothelial progenitor cells, for stimulatingvasculogenesis, for the therapy of diseases associated with adysfunction of endothelial progenitor cells and for producingpharmaceutical compositions for the treatment of such diseases, andpharmaceutical compositions which comprise erythropoietin and othersuitable active ingredients for stimulating endothelial progenitorcells.

The vascular endothelium is a layer of cells which lines blood vessels.The endothelium separates the blood from other vessel layers, and theendothelium does not just represent a passive barrier but is activelyinvolved in regulating vessel tone. Reference is also accordingly madeto endothelium-dependent vasodilatation. As a result of its position,the endothelium is permanently exposed to hemodynamic stress andmetabolic stress. Pathogenic conditions, for example elevated bloodpressure, elevated LDL levels, impaired kidney function or elevatedblood glucose, are therefore frequently associated with functionalendothelial defects which may then be followed by morphologicallydetectable lesions such as the formation of atherosclerotic plaques. Avery early sign of an altered or diminished endothelial function, orendothelial dysfunction, is a reduction in the endothelium-dependentvasodilatation.

In coronary heart disease (CHD), but also when risk factors are presentwithout CHD, for example hypertension, hyperlipoproteinemia or diabetes,the defects in endothelial function are manifested by a reducedproduction of NO (=EDRF) and increased endothelin production. Highplasma levels of endothelin lead to abnormal adhesion of cells,inflammations, vascular proliferation and severe vessel constrictions.Disturbances of endothelial function are additionally characterized byincreased production of adhesion molecules such as ICAM-1 and VCAM-1,causing platelets and monocytes to adhere to an increased extent toendothelium. The result of this is an increase in vessel tone. Thus, animbalance develops in various systems, favoring vasoconstriction,adhesion, aggregation, coagulation, atherosclerosis andatherothrombosis. Even mental stress leads to a measurable endothelialdysfunction which may persist for up to 4 hours.

Endothelial cells are involved in the formation of new blood vessels.The formation of blood vessels is important in a large number ofprocesses such as, for example, embryogenesis, the female reproductivecycle, wound healing, tumor growth and the neovascularization ofischemic areas. Originally, postnatal formation of blood vessels, thatis the formation of blood vessels after birth, was mainly attributed toangiogenic processes. Angiogenesis means the formation of new bloodvessels through capillaries sprouting from a pre-existing vascularsystem. During angiogenesis, firstly the basement membrane surroundingthe blood vessels is broken down by proteolytic enzymes, and theextracellular matrix in the perivascular space is fragmented. Theangiogenic stimuli released thereby caused differentiated endothelialcells which are already present to migrate in the direction of thechemotactic stimulus, during which they simultaneously proliferate andare transformed. Juxta-positioning of endothelial cells then forms newvessel loops with a capillary-type lumen. The onset of synthesis of anew basement membrane follows.

However, recent investigations show that the formation of new bloodvessels in the adult organism derives not only from angiogenesis butalso from vasculogenic mechanisms. Vasculogenesis means formation of newvessels from endothelial progenitor cells which are differentiating insitu. The belief that vasculogenesis is confined to embryogenesis wasrefuted by the detection of endothelial progenitor cells (EPC) inperipheral blood of healthy humans and animals. It was possible to proveby using animal models that the endothelial progenitor cells derivedfrom bone marrow are actively involved in neovascularization. It wasalso shown that a specific CD34-positive subgroup of leukocyte whichexpresses endothelium-specific antigens becomes established in ischemicregions. In addition, endothelial progenitor cells (EPC) which make asignificant contribution to the formation of blood vessels in the adultorganism can be obtained from CD133⁺ and CD34³⁰ cells in vitro (Asaharaet al., Science, 275 (1997), 964-967; Crosby et al., Circ. Res., 87(2000), 728-730; Gehling et al., Blood, 95 (2000), 3106-3112). It wasadditionally shown that injection of isolated CD34³⁰ cells or cultivatedendothelial progenitor cells expedites restoration of blood flow indiabetic mice (Schatteman et al., J. Clin. Invest., 106 (2000), 571-578)and improves neovascularization in vivo (Asahara et al., Circ. Res., 85(1999), 221-228; Crosby et al., Circ. Res., 87 (2000), 728-730; Muroharaet al., J. Clin. Invest., 105 (2000), 1527-1536). It was moreoverpossible to show that a neovascularization induced by CD34³⁰ cellsimproves cardiac function (Kocher et al., Nat. Med., 7 (2001), 430-436).

However, the mechanisms underlying the mobilization and differentiationof endothelial progenitor cells are not yet fully explained. Molecularbiological and cytobiological investigations indicate that variouscytokines and angiogenic growth factors have stimulating effects on themobilization of endothelial progenitor cells in bone marrow. Thus, it isknown that proangiogenic factors such as VEGF and GM-CSF are able toincrease the number of endothelial progenitor cells (Asahara et al.,EMBO, J., 18 (1999), 3964-3972; Takahashi et al., Nat. Med., 5 (1999),434-438). VEGF (vascular endothelial growth factor) is a protein whichoccurs in various isoforms and which binds to the two tyrosine kinasereceptors VEGF-R1 (flt-1) and VEGF-R2 (flk-1) which occur for example onthe surface of growing endothelial cells (Wernert et al., Angew. Chemie,21 (1999), 3432-3435). Activation of VEGF receptors leads via theRas-Raf-MAP kinase pathway to expression of proteinases and specificintegrins on the surface of endothelial cells or endothelial progenitorcells and finally to initiation of proliferation and migration of thesecells in the direction of the angiogenic stimulus. GM-CSF(granulocyte-macrophage colony-stimulating factor) is a cytokine whichwas previously known in particular for stimulating white bloodcorpuscles including neutrophils, macrophages and eosinophils. PIGF(placental growth factor) is known to stimulate the mobilization ofendothelial progenitor cells but not proliferation thereof.Investigations by Llevadot et al. (J. Clin. Invest., 108 (2001),399-405) reveal that HMG-CoA reductase inhibitors, especially statins,which are employed as lipid-lowering medicaments and reduce themorbidity and mortality of coronary disease, are able to mobilizeendothelial progenitor cells. Dimmeler et al. (J. Clin. Invest., 108(2001), 391-397) were able to show further that statins such asatorvastatin and simvastatin significantly improve the differentiationof endothelial progenitor cells in mononuclear cells and CD34³⁰ stemcells isolated from peripheral blood in vitro and in vivo. Thus,treatment of mice with statins led to an increased number ofdifferentiated endothelial progenitor cells, with statins showing aneffect as strong as that of VEGF.

Stimulation of the mobilization and/or differentiation of endothelialprogenitor cells represents an important novel therapeutic strategy forincreasing postnatal neovascularization, especially vasculogenesis, andfor treating diseases associated with a dysfunction of endothelialprogenitor cells and/or endothelial cells.

The present invention is based on the technical problem of providingmeans and methods for improved stimulation of endothelial progenitorcells and for the therapy of disorders particularly associated with adysfunction of endothelial progenitor cells.

The present invention solves this technical problem by disclosing theuse of erythropoietin and/or its derivatives for stimulating thephysiological mobilization of endothelial progenitor cells, theproliferation of endothelial progenitor cells, the differentiation ofendothelial progenitor cells to endothelial cells and/or the migrationof endothelial progenitor cells in the direction of an angiogenic orvasculogenic stimulus in a human or animal body. The present inventionalso solves this technical problem by disclosing the use oferythropoietin and/or its derivatives for the therapy of diseases orpathological states associated with a dysfunction of endothelialprogenitor cells and/or endothelial cells.

It has surprisingly been found according to the invention that atreatment with erythropoietin leads to physiological mobilization ofendothelial progenitor cells, with an increase in the number ofcirculating endothelial progenitor cells and induction ofdifferentiation thereof, especially in comparatively low EPO doses. Inaddition, functional deficits, which occur in certain pathologicalconditions, of the endothelial progenitor cells are compensated. It waspossible to show according to the invention that the number ofcirculating stem cells in patients with chronic kidney disease in theterminal stage is just as high as in healthy subjects, but in thesepatients they have lost the ability to differentiate to endothelialcells via endothelial progenitor cells. Thus, the number of cellscapable of adhesion and showing an endothelial cell phenotype isdistinctly reduced in patients with chronic kidney disease compared withhealthy subjects. It has now been found according to the invention thatthe number of circulating stem cells increases significantly by morethan 50% after treatment of these patients with erythropoietin. There ismoreover a drastic increase in particular in the number of cells whichdevelop an endothelial phenotype. As was demonstrated by means of afunctional cell culture assay, there is three-fold increase in theimpaired adhesion ability of the endothelial progenitor cells due toerythropoietin treatment in patients with chronic kidney disease. Theadhesion ability of differentiating endothelial progenitor cells and ofendothelial cells is one of the basic preconditions for the formation ofnew tissues and/or vessels. Erythropoietin is able in this way to induceneovascularization, in particular vasculogenesis, in tissues or organsin which corresponding vasculogenic or angiogenic stimuli are released.

Erythropoietin (called EPO hereinafter) can be used according to theinvention to stimulate the physiological mobilization of endothelialprogenitor cells, the proliferation of endothelial progenitor cells, thedifferentiation of endothelial progenitor cells to endothelial cellsand/or for migration of endothelial progenitor cells in the direction ofa vasculogenic or angiogenic stimulus in a human or animal body, inparticular an adult organism. Erythropoietin can thereforeadvantageously be employed according to the invention to stimulate theformation of new vessels by vasculogenesis in tissues or organs in whichpathological vascular lesions are present. In addition, the formation ofendothelial tissue can also be induced owing to the stimulation ofendothelial progenitor cells by erythropoietin. Erythropoietin cantherefore also be employed according to the invention for treatingdiseases of the human or animal body which are associated with adysfunction of endothelial progenitor cells and/or endothelial cells.

In connection with the present invention, “erythropoietin” or “EPO”means a substance which controls the growth or the differentiation andthe maturation of stem cells via erythroblasts to erythrocytes.Erythropoietin is a glycoprotein having 166 amino acids, threeglycosylation sites and a molecular weight of about 34 000 Da. DuringEPO-induced differentiation of erythrocyte progenitor cells there isinduction of globin synthesis and an increase in the synthesis of theheme complex and in the number of ferritin receptors. The cell can takeup more iron and synthesize functional hemoglobin thereby. Hemoglobinbinds oxygen in mature erythrocytes. Thus, erythrocytes and thehemoglobin present in them play a key role in the body's oxygen supply.These processes are initiated through the interaction of EPO with anappropriate receptor on the cell surface of erythrocyte progenitor cells(Graber and Krantz, Ann. Rev. Med. 29 (1978), 51-56).

Erythropoietin is produced mainly in the kidney, but also in smallerproportions in the liver and in the brain. Small amounts oferythropoietin are also found in the serum and, under physiologicalconditions, it is at least partly excreted in the urine. Patients withrenal failure are capable of only inadequate erythropoietin (called EPOhereinafter) production and accordingly suffer from anemia. Compensationof erythropoietin deficiency by administering erythropoietin is known.Further clinical applications of erythropoietin are in theadministration of erythropoietin for iatrogenic anemia followingchemotherapy or radiotherapy of malignant diseases or viral infections(EP 0 456 153 B1). U.S. Pat. No. 4,732,889 discloses the use oferythropoietin-containing compositions for the treatment of anemiaassociated with rheumatoid arthritis. WO 88/03808 discloses thetreatment of hemochromatosis by means of EPO-containing compositions.

The term “erythropoietin” used herein includes EPO of every origin,especially human or animal EPO. The term used herein encompasses notonly the naturally occurring, that is wild-type forms of EPO, but alsoits derivatives, analogs, modifications, muteins, mutants or others, aslong as they show the biological effects of wild-type erythropoietin.

In connection with the present invention, “derivatives” mean functionalequivalents or derivatives of erythropoietin which are obtained, withretention of the basic erythropoietin structure, by substitution of oneor more atoms or molecular groups or radicals, in particular bysubstitution of sugar chains such as ethylene glycol, and/or whose aminoacid sequences differ from that of the naturally occurring human oranimal erythropoietin protein in at least one position but essentiallyhave a high degree of homology at the amino acid level and comparablebiological activity. Erythropoietin derivatives as can be employed forexample in the present invention are disclosed inter alia in WO94/25055, EP 0 148 605 B1 or WO 95/05465.

“Homology” means in particular a sequence identity of at least 80%,preferably at least 85% and particularly preferably at least more than90%, 95%, 97% and 99%. The term “homology” which is known to the skilledworker thus refers to the degree of relationship between two or morepolypeptide molecules, which is determined by the agreement between thesequences. It is possible in this connection for an agreement to meanboth an identical agreement and a conservative amino acid exchange.

The term “derivative” also includes according to the invention fusionproteins in which functional domains of another protein are present onthe N-terminal part or on the C-terminal part. In one embodiment of theinvention, this other protein may be for example GM-CSF, VEGF, PIGF, astatin or another factor which has a stimulating effect on endothelialprogenitor cells. In a further embodiment of the invention, the otherprotein may also be a factor which has a stimulating effect ondifferentiated endothelial cells, for example angiogenin or bFGF (basicfibroblast growth factor). It is known that the growth factor bFGFexerts a strong mitogenic and chemotactic activity on endothelial cells.

The differences between an erythropoietin derivative and nativeerythropoietin may arise for example through mutations such as, forexample, deletions, substitutions, insertions, additions, base exchangesand/or recombinations of the nucleotide sequences encoding theerythropoietin amino acid sequences. Obvious possibilities in thisconnection are also naturally occurring sequence variations, for examplesequences from another organism or sequences which have been mutated ina natural way, or mutations introduced deliberately into theerythropoietin-encoding nucleic acid sequences with the aid ofconventional means known in the art, for example chemical agents and/orphysical agents. In connection with the invention, therefore, the term“derivative” also includes mutation erythropoietin molecules, that iserythropoietin muteins.

It is also possible according to the invention to employ peptide orprotein analogs of erythropoietin. In connection with the presentinvention, the term “analogs” includes compounds which do not have anamino acid sequence identical to the erythropoietin amino acid sequencebut whose three-dimensional structure greatly resembles that oferythropoietin and which therefore have a comparable biologicalactivity. Erythropoietin analogs may be, for example, compounds whichcomprise in a suitable conformation the amino acid residues responsiblefor the binding of erythropoietin to its receptors and which aretherefore able to simulate the essential surface properties of theerythropoietin binding region. Compounds of this type are described forexample in Wrighton et al., Science, 273 (1996), 458.

The EPO employed according to the invention can be produced in variousways, for example by isolation from human urine or from the urine orplasma (including serum) of patients suffering from aplastic anemia(Miyake et al., J.B.C. 252 (1977), 5558). Human EPO can be obtained forexample also from tissue cultures of human renal cancer cells (JA-OS55790/1979), from human lymphoblast cells which have the ability toproduce human EPO (JA-OS 40411/1982) and from a hybridoma cultureobtained by cell fusion of a human cell lines. EPO can also be producedby genetic engineering methods by using suitable DNA or RNA which codesfor the appropriate amino acid sequence of EPO to produce the desiredprotein recombinantly, for example in a bacterium, a yeast, a plant cellline or animal cell line. Methods of these types are described forexample in EP 0 148 605 B2 or EP 0 205 564 B2 and EP 0 411 678 B1.

The present invention relates in particular to the use of erythropoietin(called EPO hereinafter) and/or derivatives thereof to stimulate thephysiological mobilization of endothelial progenitor cells, theproliferation of endothelial progenitor cells, the differentiation ofendothelial progenitor cells to endothelial cells and/or for themigration of endothelial progenitor cells in the direction of avasculogenic or angiogenic stimulus in a human or animal body, inparticular an adult organism.

In connection with the present invention, “endothelial progenitor cells”(EPC) mean cells which circulate in the bloodstream and have the abilityto differentiate to endothelial cells. The endothelial progenitor cellswhich occur during embryonic development are angioblasts. Theendothelial progenitor cells occurring in the adult organism areangioblast-like cells which can be obtained from mononuclear cells, inparticular CD34⁻CD14⁺ monocytes, and/or CD34³⁰ stem cells, which havebeen isolated from peripheral blood.

In connection with the present invention, “mobilization” or“physiological mobilization” means the process of activating stem cellsand/or progenitor cells from the bone marrow by growth factors, withentry of the stem cells or progenitor cells into the bloodstream, inparticular into the peripheral blood.

In connection with the present invention “proliferation” means theability of cells to enlarge and subsequently divide into two or moredaughter cells. The EPO-mediated stimulation of endothelial progenitorcells thus relates in particular to the number and thus the dividingbehavior of endothelial progenitor cells.

In connection with the present invention, “Differentiation” ofendothelial progenitor cells means the development of mononuclear cellsderived from the bone marrow via endothelial progenitor cells intoendothelial cells. “Endothelial cells” mean the cells which form theendothelium, that is the monolayer cellular lining of vessels and serouscavities. Endothelial cells are characterized in that they releasevasoactive substances, for example vasodilating substances such as EDRF(endothelial derived relaxing factor) or constricting substances such asendothelin, factors for inhibition or activation of blood clotting andfactors for regulating vascular permeability. Endothelial cells alsosynthesize components of the subendothelial connective tissue,especially collagens of type IV and V, cell adhesion proteins such aslaminin, fibronectin and thrombospondin, growth factors, for example forsmooth muscle cells, and factors for the formation of new vessels.

In connection with the present invention, “migration” of endothelialprogenitor cells means that the endothelial progenitor cells present inthe bloodstream migrate in the direction of a vasculogenic or angiogenicstimulus and become concentrated in the region of the vasculogenic orangiogenic stimulus. A “vasculogenic stimulus” means a chemical stimulusin a tissue or blood vessel of a human or animal body which actsspecifically on endothelial progenitor cells and brings about migrationthereof to the site in the body from which the chemical stimulusoriginates. The vasculogenic stimulus induces in this way thevasculogenesis process. An “angiogenic stimulus” means a chemicalstimulus in a tissue or blood vessel of a human or animal body whichacts specifically on differentiated endothelial cells and brings aboutmigration thereof to the site in the body from which the chemicalstimulus originates. The angiogenic stimulus brings about in this way aninduction of angiogenesis.

A further embodiment of the invention provides the use of erythropoietinand/or derivatives thereof for increasing the adhesion ability ofdifferentiating endothelial progenitor cells. Erythropoietin is usedaccording to the invention in particular for improving the adhesionability of endothelial progenitor cells, that is for cell-cell adhesion.The adhesion of differentiating endothelial progenitor cells ordifferentiated endothelial cells is one of the basic preconditions forthe formation of new vessels or of new endothelial tissue. Cell adhesionis mediated by protein molecules.

The present invention also relates to the use of erythropoietin forstimulating the formation of new vessels, in particular stimulation ofvasculogenesis. In connection with the present invention,“vasculogenesis” means the formation of new vessels from endothelialprogenitor cells which are differentiating in situ. Thus, according tothe invention, the use of erythropoietin results in increasedinvolvement of endothelial progenitor cells in the formation of newvessels or in a local formation of new vessels to restore damagedvascular regions. The invention thus provides for the use oferythropoietin and/or its derivatives to promote the formation of newblood vessels and/or the replacement of damaged vascular regions throughlocal formation of new blood vessels.

A further embodiment of the invention provides for the use oferythropoietin and/or derivatives thereof for stimulating endothelialprogenitor cells to form endothelial tissue.

A particularly preferred embodiment of the invention provides the use oferythropoietin and/or derivatives thereof for the therapy ofpathological states or diseases of the human or animal body which areassociated with a dysfunction of endothelial progenitor cells, or ofsequelae thereof.

In connection with the present invention, “diseases”, “pathologicalstates” or “disorders” mean disturbances of vital processes in organs orin the whole body resulting in subjectively experienced or objectivelydetectable physical, mental or intellectual changes. The invention isconcerned in particular with diseases associated with a dysfunction ofendothelial progenitor cells, that is diseases which either are theresult of such a dysfunction of these cells or are mediated by thesecells. “Sequelae” mean secondary diseases, that is a second disorderadded to a primary pathological state.

In connection with the present invention, a “dysfunction” of endothelialprogenitor cells means a disturbance of essential cell functions such asmetabolic activities, response to stimuli, motility, dividing behavioror differentiation behavior of these cells. A dysfunction of endothelialprogenitor cells may consist for example of only inadequate or noproliferation of these cells. Since the proliferation of endothelialprogenitor cells is stimulated by the use of erythropoietin, it is thuspossible to compensate the deficient dividing behavior both ofendothelial progenitor cells and of previously differentiatedendothelial cells, and to increase the number of endothelial progenitorcells or endothelial cells. A dysfunction of endothelial progenitorcells may consist for example of the impaired ability of these cells todifferentiate to endothelial cells. The dysfunction of endothelialprogenitor cells may also be caused by their impaired adhesion abilityand/or their impaired ability to migrate in the direction of anangiogenic or vasculogenic stimulus. Such dysfunctions of endothelialprogenitor cells may lead for example to the impairment or prevention ofthe formation of new endothelial tissue and/or vasculogenesis. Adysfunction of endothelial progenitor cells may also have a pathogeniccause, for example due to hypertension, hyperlipoproteinemia, uremia ordiabetes. The dysfunction of endothelial progenitor cells may bemanifested for example by a reduced production of NO (=EDRF) by NOsynthases (NOS) from L-arginine, increased endothelin production and/orenhanced production of adhesion molecules such as ICAM-1 and VCAM-1.

The diseases associated with a dysfunction of endothelial progenitorcells are according to the invention in particular hypercholesterolemia,diabetes mellitus, endothelium-mediated chronic inflammatory disorderssuch as inflammations of vessels, endotheliosis includingreticuloendotheliosis, atherosclerosis, coronary heart disease,myocardial ischemia, angina pectoris, age-related cardiovasculardisorder, ischemic disorders of the extremities, Raynaud's disease,preeclampsia, pregnancy-induced hypertension, chronic or acute renalfailure, especially terminal renal failure, heart failure, wound healingand sequelae thereof.

“Hypercholesterolemia” is characterized by elevated concentrations ofcholesterol in the blood. By far the commonest form of primaryhypercholesterolemia is polygenic hypercholesterolemia. Secondaryhypercholesterolemias frequently occur in association with diabetesmellitus, nephrotic syndrome, hypothyroidism and hepatic disorders.

“Diabetes mellitus” encompasses various forms of glucose metabolismdisorders differing in etiology and symptoms. Responsible for thedevelopment of vessel-related diabetic complications is, in particular,the AGE-RAGE system. AGEs (advanced glycation endproducts) are producedby a series of complex reactions after long-lasting exposure of proteinsor lipids to reducing sugars, for example glucose. The formation of AGEstakes place during the normal aging process and to an increased extentin diabetes mellitus and Alzheimer's disease. Binding of AGEs leads tooxidative stress, activation of the NF-κB transcription factor and thusa disturbance of endothelial homeostasis.

“Endothelium-mediated chronic inflammatory disorders” are disorders orconditions of a human or animal body which derive from a defenseresponse of the body and its tissues to harmful stimuli, with certainsignal molecules altering the properties of endothelial cells so that,in concert with the activation of other cell types, leukocytes remainadherent to endothelial cells, finally penetrate into the tissue andthere initiate inflammation. One example of an endothelium-mediatedinflammation is leukocytic vasculitis. A central part is played in theactivation of an endothelium-mediated inflammatory event by thetranscription factor NF-κB. Another system leading to the development ofendothelial cell-mediated chronic inflammations is the AGE-RAGE system.

“Endotheliosis” means degenerative and proliferative endothelial changesassociated with non-thrombopenic purpura. “Reticuloendotheliosis” meansdiseases of the reticulohistiocytic system, such as reticulum,reticulosis, reticulohistiocytosis and Hand-Schüller-Christian disease.

“Myocardial ischemia” means bloodlessness or hypoperfusion, that is animpairment of the blood supply, of the muscular wall of the heart as aresult of inadequate or absent arterial supply of blood. A “cardiacinfarct” or “myocardial infarct” is a necrosis of a localized region ofthe myocardium, which usually occurs as an acute event complicatingchronic coronary heart disease. “Coronary heart disease” or “ischemicheart disease” is a degenerative coronary disorder which, owing to aconstriction or a closure of coronary vessels of the heart, leads to areduced blood supply to the myocardium. “angina pectoris” means an acutecoronary insufficiency or stenocardia which may be induced by animbalance of the oxygen supply and oxygen demand associated withcoronary heart disease, coronary spasms, impairments of blood flow,cardiac arrhythmias, hypertension or hypotension. “Raynaud's disease”means ischemic states which are caused by vasoconstriction, that isvessel spasms, and occurs episodically, usually in the arteries of thefingers. Primary Raynaud's disease is a purely functional impairment ofthe small vessels supplying the distal parts of the extremities, whereassecondary Rynaud's disease has another disease underlying it, forexample an inflammation of vessels.

“Preeclampsia” is an endothelial and vascular disease of the maternalbody and appears to be the effect of endotheliotropic substances fromthe placenta. Preeclampsia is a multisystem disorder which may lead todisturbances of function of numerous organs and be manifested by diversesymptoms. The impairments of blood supply which are typical of thedisorder are the result of an increased vascular resistance, possiblywith local variations in severity. It is regarded as confirmed that anendothelial dysfunction is the central component of the pathogenesis ofpreeclampsia.

“Renal failure” means in connection with the present invention therestricted ability of the kidneys to excrete substances normallyexcreted in the urine, and in advanced stages there is also loss of theability to regulate the electrolyte, water and acid-base balance.Terminal renal failure is characterized by a collapse of the excretoryand endocrine function of the kidneys.

The renal failure may according to the invention be acute renal failurewhich is also referred to as acute kidney failure or shock aneuria.Acute renal failure is characterized by a sudden partial or completeloss of the excretory function of the kidneys as a result of usuallyreversible kidney damage. The cause may be hypoperfusion due tohypovolemia, hypotension and dehydration resulting from blood losses(polytrauma, gastrointestinal or postpartum bleeding, major surgicalprocedures on the heart, vessels, abdomen or prostate), shock(myocardial infarct, embolism), serious infections (sepsis, peritonitis,cholecystitis), hemolysis (hemolytic-uremic syndrome, paroxysmalhemoglobulinuria, transfusion reaction), myolysis (crush syndrome,rhabdomyolysis, myositis, burns), water and electrolyte losses (massivevomiting, diarrhea, excessive sweating, ileus, acute pancreatitis).Further causes may be nephrotoxins such as exogenous toxins, for exampleaniline, glycol compounds, methanol and the like, or endogenous toxins,for example myoglobin and oxalates. Further causes of acute renalfailure are renal diseases, for example inflammatory nephropathies orrejection reactions following renal transplantation. Acute renal failuremay also be caused by retention of urine following obstruction to theflow of urine. The treatment according to the invention of acute renalfailure with erythropoietin leads according to the invention toprevention or at least diminution of progression of acute renal failure.

The renal failure may according to the invention also be chronic renalfailure. Causes of chronic renal failure are vascular, glomerular andtubulointerstitial renal disorders, infections and inborn or acquiredstructural defects. Causes of chronic renal failure are, inter alia,chronic glomerulopathy, chronic pyelonephritis, analgesic nephropathy,obstructive uropathy and arterio- and arteriolosclerosis. The terminalstage of chronic renal failure is uremia. The treatment according to theinvention of chronic renal failure with erythropoietin leads accordingto the invention to a diminution in the progression of chronic renalfailure.

In connection with the present invention, “heart failure” means apathological state which is also referred to as myocardial insufficiencyor weakness of the heart muscle. Heart failure is characterized byinadequate functioning of the heart, the heart no longer being capableof efficient delivery to comply with the requirements. Heart failure canbe categorized according to various aspects. For example, according tothe affected segment of the heart it is classified as right heartfailure, left heart failure and failure on both sides (global failure).According to the stability of an equilibrium influenced by physiologicaland therapeutic mechanisms, a distinction is made between compensatedand decompensated heart failure. Classification takes place into acuteand chronic heart failure according to the time course. Causes of heartfailure are, inter alia, myocardial infarction, cardiomyopathy, inbornor acquired cardiac defects, essential or pulmonary hypertension,cardiac arrhythmias, coronary heart disease or myocarditis.

In connection with the present invention, “wound healing” means thephysiological processes for regenerating damaged tissue and for closinga wound, especially formation of new connective tissue and capillaries.The wound healing may be primary wound healing (first intentionhealing), which is characterized by rapid and complication-free closureand substantially complete recovery as a result of minimal formation ofnew connective tissue between the edges of a wound, which have a goodblood supply and are approximated where appropriate, of a clean wound.Wounds where the edges of the wound are further apart and, inparticular, crushed or necrotic, and infected wounds, undergo delayedsecondary wound healing (second intention healing) in which, as a resultof an (a)bacterial inflammation, there is filling of the tissue defectwith granulation tissue and extensive formation of scar tissue.Epithelialization starting from the edge terminates the wound healing.The wound healing is divided into three phases, namely latency phase,proliferative phase and repair phase. The latency phase in turn isdivided into the oxidative phase with scab formation, especially in thefirst few hours after the wound occurred, and the absorptive phase withcatabolic autolysis, which extends over a period of from one to threedays after the wound occurred. The proliferative phase is characterizedby anabolic repair with production of collagen by fibroblasts and occurson the fourth to seventh day after the wound occurred. The repair phaseoccurs after the eighth day after the wound occurred and ischaracterized by transformation of the granulation tissue into a scar.

A “wound” means in connection with the present invention an interruptionof the coherence of body tissues with or without loss of substance andcaused by mechanical injury or physically caused cell damage. Types ofwound are mechanical wounds, thermal wounds, chemical wounds, radiationwounds and disease-related wounds. Mechanical wounds arise throughtraumatic violence and occur in particular as incision and puncturewounds, crushing, lacerating, tearing and abrading wounds, scratch andbite wounds and projective wounds. Thermal wounds arise through exposureto heat or cold. Chemical wounds arise in particular through the actionof acids or alkalis. Radiation wounds arise for example through exposureto actinic and ionizing radiation. Wounds occurring in relation todisease are in particular congestion-related wounds, traumatic wounds,diabetic wounds etc. The invention provides in particular forerythropoietin to be administered preferably topically or intravenouslyfor wound healing.

The present invention therefore relates to the use of erythropoietin forthe therapy of hypercholesterolemia, diabetes mellitus,endothelium-mediated chronic inflammatory disorders, endotheliosisincluding reticuloendotheliosis, atherosclerosis, coronary heartdisease, myocardial ischemia, angina pectoris, age-relatedcardiovascular disorders, ischemic disorders of the extremities,preeclampsia, Raynaud's disease, pregnancy-induced hypertension, chronicor acute renal failure, especially terminal renal failure, heartfailure, wound healing and sequelae thereof.

The invention provides for erythropoietin to be administered to apatient in a therapeutically effective dose which is sufficient to curethe condition of an aforementioned disease, in particular a diseaseassociated with a dysfunction of endothelial progenitor cells, or toprevent this condition, to stop the progression of such a disease and/orto alleviate the symptoms of such a disease. The dose to be administeredto a patient depends on many factors, for example the age, body weightand gender of the patient, the severity of the disorders etc.

It is particularly preferred according to the invention forerythropoietin, in all the uses, methods and compositions of the presentdisclosure, to be used in very small amounts which are below the amountsknown to be employed, administering in particular in vivo, i.e. perpatient, EPO doses of from 200 to 2 000 units (IU; internationalunits)/week, preferably doses of from 500 to 2 000 IU/week, depending onthe severity of the disorder and depending on renal function. The doses,provided according to the invention, of from 200 to 2 000 units(IU)/week and per patient, especially from 500 to 2 000 IU/week and perpatient, are subpolycythemic doses, that is doses which do not lead toerythrocytes with hematocrit values of more than 50%. Thesubpolycythemic doses provided according to the invention correspond toweekly doses of about 1 to 90 units (IU) of EPO/kg of body weight, inparticular 1 to 45 IU of EPO/kg of body weight, or a comparable weeklydose of Aranesp of from 0.005 to 0.45 μg/kg of body weight, inparticular 0.005 to 0.225 μg/kg of body weight. Aranesp is a doublyPEGylated EPO. The dose of from 200 to 2 000 units/week per patient, inparticular from 500 to 2 000 IU/week and per patient, which is providedaccording to the invention for the treatment of diseases or pathologicalstates associated with a dysfunction of endothelial progenitor cells isvery low compared with the initial dose of 50-150 IU/kg of bodyweight/week (usually starting with 4 000-8 000 IU/week, but alsoconsiderably higher if the result of therapy is unsatisfactory) normallyemployed for the therapy of renal anemia.

A particularly preferred embodiment of the invention relates to the useof erythropoietin and/or its derivative as active ingredient forproducing a pharmaceutical composition or a medicament for the therapyof pathological states or diseases associated with a dysfunction ofendothelial progenitor cells.

An “active ingredient” means according to the invention an endogenous orexogenous substance which on contact with target molecules or targetcells or target tissues influences in a differentiated manner specificfunctions of tissues, organs or organisms. The invention thus providesfor erythropoietin as active ingredient of the pharmaceuticalcomposition of the invention influencing the proliferation,differentiation and/or migration behavior of endothelial progenitorcells on contact therewith in a human or animal organism in such a waythat dysfunctions of endothelial progenitor cells can be compensated andthe diseases occurring as a consequence of these dysfunctionseffectively controlled, alleviated or eliminated, or these diseaseseffectively prevented.

In connection with the present invention, a “pharmaceutical composition”or a “medicament” means a mixture which is used for diagnostic,therapeutic and/or prophylactic purposes, that is promoting or restoringthe health of a human or animal body, and which includes at least onenatural or synthetically produced active ingredient which brings aboutthe therapeutic effect. The pharmaceutical composition may be either asolid or a liquid mixture. For example, a pharmaceutical compositionincluding the active ingredient may comprise one or morepharmaceutically acceptable components. The pharmaceutical compositionmay additionally include additives normally used in the art, for examplestabilizers, manufacturing materials, release agents, disintegrants,emulsifiers or other substances normally used for pharmaceuticalcomposition production.

The invention provides in particular the use of erythropoietin and/or aderivative thereof as active ingredient for producing a medicament forthe therapy of hypercholesterolemia, diabetes mellitus,endothelium-mediated chronic inflammatory disorders such asinflammations of vessels, endotheliosis including reticuloendotheliosis,atherosclerosis, coronary heart disease, myocardial ischemia, anginapectoris, age-related cardiovascular disorder, ischemic disorders of theextremities, Raynaud's disease, preeclampsia, pregnancy-inducedhypertension, acute or chronic renal failure, especially terminal renalfailure, heart failure, wound healing and sequelae thereof.

The pharmaceutical composition of the invention may be suitable both fortopical and for systemic administration.

A preferred embodiment of the invention provides for the pharmaceuticalcomposition to be used for parenteral, in particular intravenous,intramuscular, intracutaneous or subcutaneous administration. Theerythropoietin-containing medicament preferably has the form of aninjection or infusion.

A further use provides for the erythropoietin-containing pharmaceuticalcomposition to be administered orally. For example, theerythropoietin-containing medicament is administered in a liquid dosageform such as a solution, suspension or emulsion, or a solid dosage formsuch as a tablet.

A further use provides for the pharmaceutical composition to be suitablefor pulmonary administration or for inhalation. The invention thusprovides for erythropoietin to be administered in a therapeuticallyeffective manner directly onto the lungs of the patient. This type ofadministration of erythropoietin makes it possible to deliver anerythropoietin dose quickly to a patient without the need to perform aninjection. When erythropoietin is absorbed through the lungs it ispossible to deliver considerable amounts of erythropoietin via the lungsto the bloodstream, which leads to increased amounts of erythropoietinin the bloodstream. In a preferred embodiment of the invention, thepharmaceutical composition to be absorbed through the lung is an aqueousor nonaqueous solution or a dry powder. When theerythropoietin-containing medicament to be administered by the pulmonaryroute is in the form of a dry powder, the latter preferably includeserythropoietin-containing particles, where the particles have a diameterof less than 10 μm, so that the medicament can also reach distal regionsof the patient's lung. A particularly preferred embodiment of theinvention provides for the medicament which is to be administered by thepulmonary route to be in the form of an aerosol.

A particularly preferred embodiment of the invention relates to the useof erythropoietin for producing a pharmaceutical composition for thetherapy of diseases associated with a dysfunction of endothelialprogenitor cells, where the pharmaceutical composition comprises besideserythropoietin as active ingredient at least one further additionalactive ingredient to stimulate endothelial progenitor cells.

The further active ingredient is preferably an active ingredient whichstimulates in particular the physiological mobilization of endothelialprogenitor cells from the bone marrow. However, the further activeingredient may also be according to the invention an active ingredientwhich stimulates in particular the dividing behavior, that is theproliferation, of endothelial progenitor cells. However, there is alsothe possibility according to the invention for the further activeingredient in particular to stimulate the differentiation behaviorand/or the migration behavior of endothelial progenitor cells. Thefurther active ingredient which stimulates endothelial progenitor cellsis particularly preferably VEGF, PIGF, GM-CSF, an HMG-CoA reductaseinhibitor, in particular a statin such as simvastatin, mevastatin oratorvastatin, and/or an NO donor, especially L-arginine.

The invention also provides for the at least one further activeingredient in particular to stimulate differentiated endothelial cells,that is the proliferation and/or migration thereof, but not endothelialprogenitor cells. Particular preference is given in this connection tobFGF (basic fibroblast growth factor) or angiogenin.

A further embodiment of the invention relates to the use oferythropoietin and/or derivatives thereof as active ingredient forproducing a pharmaceutical composition to stimulate endothelialprogenitor cells, in particular to stimulate the mobilization,proliferation, differentiation to endothelial cells and/or for migrationin the direction of a vasculogenic or angiogenic stimulus. The inventionfurther provides for the use of erythropoietin and/or its derivatives asactive ingredient for producing a pharmaceutical composition tostimulate vasculogenesis and/or endothelium formation, in particular inthe adult human or animal organism.

The present invention therefore also relates to pharmaceuticalcompositions to stimulate endothelial progenitor cells, in particular tostimulate the mobilization, proliferation, differentiation thereof toendothelial cells and/or migration in the direction of a vasculogenic orangiogenic stimulus, to stimulate vasculogenesis and/or endotheliumformation and for the treatment of disease of the human or animal bodywhich are associated with a dysfunction of endothelial progenitor cellsand/or endothelial cells. The present invention relates in particular topharmaceutical compositions or medicaments which include erythropoietinas active ingredient and at least one further active ingredient tostimulate endothelial progenitor cells and/or differentiated endothelialcells. In a preferred embodiment, the present invention relates topharmaceutical compositions which include erythropoietin and at leastone further active ingredient from the group consisting of VEGF, PIGF,GM-CSF, an HMG-CoA reductase inhibitor, in particular a statin such assimvastatin, mevastatin or atorvastatin, an NO donor, especiallyL-arginine, bFGF and angiogenin.

A further preferred embodiment of the invention relates to the use oferythropoietin for producing a transplantable endothelial cellpreparation. The invention provides in this connection in particular forendothelial cells to be produced in vitro by cultivating endothelialprogenitor cells in the presence of erythropoietin and subsequentlytransplanted into a recipient organism, in particular an organismsuffering from a disease associated with a dysfunction of endothelialprogenitor cells. For example, mononuclear cells (MNC) can be isolatedfrom blood by density gradient centrifugation and cultivated in suitableculture media in vitro. Methods for the isolation and in vitrocultivation of mononuclear cells are described for example in Asahara,Science, 275 (1997), 964-967; Dimmeler et al., J. Clin. Invest., 108(2001), 391-397 and Llevadot et al., J. Clin. Invest., 108 (2001)399-405. The mononuclear cells are then cultivated further in thepresence of erythropoietin in order to stimulate the proliferation anddifferentiation behavior of the endothelial progenitor cells present inthe MNCs, and in particular to increase the number of differentiatedadherent endothelial cells. The invention also provides for thecultivation of the MNCs to take place in the presence of erythropoietinand at least one further substance which stimulates the proliferationand differentiation of endothelial progenitor cells. The furthersubstance particularly preferably employed is VEGF, PIGF, GM-CSF, an NOdonor such as L-arginine or an HMG-CoA reductase inhibitor such as astatin, in particular simvastatin, mevastatin or atorvastatin.

A further preferred embodiment of the invention provides for the use oferythropoietin for the pretreatment and/or further treatment of tissuesor organs to be transplanted. In this case, the transplants are treatedwith erythropoietin before the transplantation, preferably immediatelybefore, while still in the donor organism. The recipient organism canlikewise be treated with erythropoietin from the time of transplantationonwards. This treatment of the organs or tissues to be transplanted,both directly before and after transplantation, with erythropoietinachieves according to the invention rapid formation of new blood vesselsin the transplant after transplantation has taken place into a body,because of the induced vasculogenesis, and rapid connection of thesenewly formed blood vessels to the blood system of the recipientorganism. The formation of endothelia is likewise achieved quickly inthis way. The treatment of organ or tissue transplants witherythropoietin thus brings about faster incorporation of these systemsin the body, thus considerably reducing the risk of rejection.

A further development of the invention provides for the organ or tissuetransplants to be treated before transplantation with erythropoietin incombination with at least one further factor which stimulatesendothelial progenitor cells. This factor is preferably a substance fromthe group consisting of VEGF, PIGF, GM-CSF, an HMG-CoA reductaseinhibitor, for example a statin, in particular simvastatin, mevastatinor atorvastatin, or an NO donor, in particular L-arginine. A furtherdevelopment provides for the organ or tissue transplants to be treatedbefore transplantation besides erythropoietin with a further substancewhich stimulates the proliferation and migration of differentiatedendothelial cells. This substance is particularly preferably angiogeninor bFGF. A further development provides for the pretreatment of theorgan or tissue transplants with erythropoietin to take place usingisolated and, where appropriate, in vitro expanded endothelialprogenitor cells.

A further particularly preferred embodiment of the invention providesfor erythropoietin to be used to produce implantable or transplantablecell-containing in vitro organs or tissues. The invention provides inparticular for the organ or tissue produced in vitro to be treatedbefore the transplantation or implantation with erythropoietin in vitroin order to stimulate endothelial progenitor cells which are present inthe body of the recipient organism, especially the physiologicalmobilization, migration, proliferation and differentiation thereof. Therecipient organism is preferably treated further, after transplantationor implantation of the in vitro organ or tissue, with erythropoietin inthe doses of the invention. Treatment of the in vitro organ or tissuebefore transplantation or implantation with erythropoietin and, whereappropriate, subsequent treatment of the recipient organism witherythropoietin achieves according to the invention rapid formation ofnew blood vessels in the in vitro organ or tissue system aftertransplantation or implantation has taken place into a body, because ofthe induced vasculogenesis, and rapid connection of these newly formedblood vessels to the blood system of the recipient organism. Rapidformation of endothelia and thus reendothelialization is likewiseachieved in this way. Treatment of the in vitro organ or tissue systemswith erythropoietin thus brings about faster incorporation of thesesystems into the body, thus considerably reducing the risk of rejection,and serves to protect the transplant.

An “in vitro organ or tissue system” means a transplantable orimplantable cell-containing tissue or organ which is produced in vitrousing defined cells and/or defined tissues and under defined cultureconditions. An “implantable in vitro organ or tissue system” means asystem which, besides cells, includes exogenous materials. A“transplantable in vitro organ or tissue system” means in particular acell-containing system which, besides cells, tissue or organs of thesame or a different individual, comprises endogenous substances. Invitro organs or tissues are characterized in particular by substantiallycorresponding in terms of their structure to the native organs ortissues which are to be replaced, and thus are able to undertake thefunction of the replaced native organs or tissues in vivo.

One development of the invention provides for the in vitro organ ortissue systems to be treated before transplantation or implantation witherythropoietin in combination with at least one further factor whichstimulates endothelial progenitor cells. This factor is preferably asubstance from the group consisting of VEGF, PIGF, GM-CSF, an HMG-CoAreductase inhibitor, in particular simvastatin, mevastatin oratorvastatin, and an NO donor. A further development provides for the invitro organ or tissue systems to be treated before transplantation orimplantation besides erythropoietin with a further substance whichstimulates the proliferation and migration of differentiated endothelialcells. This substance is particularly preferably angiogenin or bFGF. Afurther development provides for the in vitro organ or tissue systemsadditionally to comprise isolated and, where appropriate, in vitroexpanded endothelial progenitor cells.

A further preferred embodiment of the invention relates to the use oferythropoietin for producing vascular prostheses or heart valves, wherethe vascular prostheses or heart valves are coated with erythropoietinbefore insertion into a body, in particular a human body. The coating ofthe vascular prostheses or heart valves with erythropoietin achievesstimulation of endothelial progenitor cells in the body of the recipientorganism, stimulating in particular their mobilization from the bonemarrow, their proliferation, their differentiation to endothelial cellsand their migration to the employed vascular prostheses or heart valves.Following introduction of the vascular prosthesis or heart valvesproduced in this way into a body, the latter can be treated further witherythropoietin, in particular in the doses of the invention. Thisresults in faster formation of endothelial layers on the employedvascular prostheses and thus faster incorporation into the relevant areaof the body. A preferred development provides for additionally employedisolated and, where appropriate, in vitro expanded endothelialprogenitor cells for coating the vascular prostheses and heart valves.

The present invention likewise relates to a method for stimulatingendothelial cell formation in vitro comprising

-   -   a) isolation of cell populations comprising endothelial        progenitor cells from blood by means of density gradient        centrifugation    -   b) cultivation of the isolated cell populations comprising        endothelial progenitor cells in cell culture medium, and    -   c) cultivation of the cell populations in the presence of        erythropoietin.

The cultivation of the cell populations can according to the inventiontake place in the presence of a further substance which stimulatesendothelial progenitor cells.

The present invention further relates to a method for treating diseasesassociated with a dysfunction of endothelial progenitor cells byadministering erythropoietin in a dose of from 200 to 2 000 IU/week, inparticular in a dose of from 500 to 2 000 IU/week, to a patient withsuch a disease. The method of the invention is particularly suitable fortreating diseases of the human body such as hypercholesterolemia,diabetes mellitus, endothelium-mediated chronic inflammatory disorderssuch as inflammations of vessels, endotheliosis includingreticuloendotheliosis, atherosclerosis, coronary heart disease,myocardial ischemia, angina pectoris, age-related cardiovasculardisorder, ischemic disorders of the extremities, Raynaud's disease,preeclampsia, pregnancy-induced hypertension, acute or chronic renalfailure, especially terminal renal failure, heart failure, wound healingand sequelae.

A preferred embodiment of the method of the invention for treatingdiseases associated with a dysfunction of endothelial progenitor cellsprovides for administration to the patient besides erythropoietin of atleast one further active ingredient selected from the group consistingof VEGF, PIGF, GM-CSF, an HMG-CoA reductase inhibitor and an NO donor.The HMG-CoA reductase inhibitor which is administered is preferably astatin such as simvastatin, mevastatin or atorvastatin. The NO donorwhich is administered is preferably L-arginine.

A further preferred embodiment of the method of the invention fortreating diseases associated with a dysfunction of endothelialprogenitor cells provides for endothelial progenitor cells to beisolated from the blood of a human organism, to be expanded in vitrousing erythropoietin and to be differentiated to endothelial cells and,after purification and isolation of the differentiated endothelial cellsor the differentiating endothelial progenitor cells, the latter then tobe transplanted in a targeted manner into a region of the body, a tissueor an organ of a patient which is damaged owing to the dysfunction ofendothelial progenitor cells and/or endothelial cells, in order toinduce local formation of new endothelium there. A more targeted andfaster treatment of the damaged regions of the body, tissues and/ororgans of the patient is possible in this way. This embodiment of themethod of the invention for treating diseases associated with adysfunction of endothelial progenitor cells comprises the followingsteps:

-   -   a) isolation of cell populations comprising endothelial        progenitor cells from blood by means of density gradient        centrifugation,    -   b) cultivation of the cell populations comprising endothelial        progenitor cells in cell culture medium,    -   c) cultivation of the cell populations comprising endothelial        progenitor cells in the presence of erythropoietin to stimulate        the proliferation of endothelial progenitor cells and/or        differentiation thereof to endothelial cells,    -   d) isolation and purification of the differentiated endothelial        cells, and    -   e) transplantation of the differentiated endothelial cells into        a body with a disease associated with a dysfunction of        endothelial progenitor cells.

Following transplantation of the differentiated endothelial cells into abody, the latter can be treated further with erythropoietin, inparticular in the doses of from 200 to 2 000 IU/week provided accordingto the invention.

It is possible according to the invention for the cell populationscomprising endothelial progenitor cells to be cultivated in vitro in thepresence of at least one further active ingredient selected from thegroup consisting of VEGF, PIGF, GM-CSF, an HMG-CoA reductase inhibitorand an NO donor. The HMG-CoA reductase inhibitor used for thecultivation is preferably a statin such as simvastatin, mevastatin oratorvastatin.

A further preferred embodiment of the invention relates to a method fortreating vascular disorders, comprising:

-   -   a) isolation of cell populations comprising endothelial        progenitor cells from blood by means of density gradient        centrifugation,    -   b) cultivation of the cell populations comprising endothelial        progenitor cells in cell culture medium,    -   c) cultivation of the cell populations comprising endothelial        progenitor cells in the presence of erythropoietin to stimulate        the proliferation of endothelial progenitor cells and/or        differentiation thereof to endothelial cells,    -   d) isolation and purification of the differentiated endothelial        cells, and    -   e) transplantation of the endothelial cells into a body with a        vascular disorder.

Following transplantation of the endothelial cells into the body with avascular disorder, the latter can be treated further witherythropoietin, preferably in the doses of the invention of from 200IU/week to 2 000 IU/week.

It is possible according to the invention for the cell populationscomprising endothelial progenitor cells to be cultivated in the presenceof at least one further active ingredient selected from the groupconsisting of VEGF, PIGF, GM-CSF and/or an HMG-CoA reductase inhibitor.The HMG-CoA reductase inhibitor used for the cultivation is preferably astatin such as simvastatin, mevastatin or atorvastatin.

The method of the invention for treating vascular disorders thusprovides for endothelial progenitor cells to be isolated from the bloodof a human organism, to be expanded in vitro using erythropoietin and tobe differentiated to endothelial cells and, after purification andisolation of the differentiated endothelial cells or the differentiatingendothelial progenitor cells, the latter then to be transplanted in atargeted manner into a damaged blood vessel or an ischemic region inorder to induce local neovascularization there. More targeted and fastertreatment of damaged blood vessels or ischemic tissues is possible inthis way. The method of the invention for treating vascular disorders isparticularly suitable for treating vascular disorders such as ischemia,especially cerebral ischemia, ischemic disorders of the extremities,myocardial ischemia, myocardial infarction, stroke, coronary heartdisease, angina pectoris, acute arterial occlusion, arterial occlusivedisease, Raynaud's disease and ergotism.

Further advantageous developments of the invention are evident from thedependent claims.

The invention is explained in more detail by means of the followingfigures and examples.

FIG. 1 shows the results of a FACS analysis of circulating CD34³⁰ stemcells (cSC). (A-D): patients' samples; (E-F): isotype controls. cSC wereidentified by means of the additional expression of the CD34 marker (Band F), by means of the characteristic low to moderate CD45 antigenexpression (C and G) and by means of the characteristic light scatteringproperties (D and H). The absolute cSC number was calculated per 100 000monocytes and lymphocytes.

FIG. 2 shows a quantitative determination of circulating stem cells bymeans of flow cytometry. The figure shows the time-dependent effect oferythropoietin treatment using rhEPO (recombinant human erythropoietin)after 0, 2, 4, 6 and 8 weeks. n=11, the values correspond to averages+/− standard deviation. Medians depicted as line.

-   *: p<0.01 compared with 2 weeks; □.□.p<0.05 compared with 4 weeks,    #: p<0.05 compared with 8 weeks.

FIG. 3 shows a quantitative determination of cultivated endothelialprogenitor cells (EPC). The figure shows that rhEPO treatment increasesthe relative number of EPCs. EPCs were isolated before the treatment ofrenal patients with rhEPO and 2, 4, 6 and 8 weeks after treatment of thepatients with rhEPO, and characterized by means of their adhesionability and the two markers acLDL-Dil and UEA-1 FITC. n=11, the valuescorrespond to averages +/− standard deviation. Medians depicted as line.

-   *: p<0.01 compared with the period before treatment;-   #: p<0.001 compared with the period before treatment.

FIG. 4 shows the quantitative determination of cultivated endothelialprogenitor cells (EPC). The figure shows that the absolute number ofEPCs before initiation of rhEPO therapy is significantly reducedcompared with healthy age- and gender-matched subjects. Patients withrenal anemia thus show distinct EPC dysfunction compared with controlsubjects. This reduced number of functional EPC was compensated 8 weeksafter starting rhEPO therapy for renal anemia. EPCs were isolated beforethe treatment of renal patients with rhEPO and 2, 4, 6 and 8 weeks aftertreatment of the patients with rhEPO, and characterized by means oftheir adhesion ability and the two markers acLDL-Dil and UEA-1 FITC.n=11. The example shown is the course over 8 weeks and all the controls.The absolute values are shown on the one hand as individual values. Inaddition, box plots are shown (90th/75th/50th/25th and 10th percentilesand the average). Age- and gender-matched subjects for which EPCs wereisolated and characterized analogously (n=11) served as healthy control.

FIG. 5 shows the effect of erythropoietin on wound healing. The figureshows that on treatment of a standardized skin wound on mice, made witha tissue punch, with erythropoietin the wound was completely closed onlyafter 7 to 8 days, whereas on treatment of the wound with physiologicalsodium chloride solution (saline) the wound was not completely closeduntil after 13 to 14 days. Treatment with erythropoietin orphysiological sodium chloride solution started 7 days before making theskin wound. Recombinant human erythropoietin was administered once aweek by s.c. (subcutaneous) injection (0.1 μg/kg Aranesp) (n=5 pergroup).

FIG. 6 shows that erythropoietin diminishes the loss of renal functionafter acute kidney failure (acute renal failure). Sprague Dawley rats(250-300 g) were included in the study. The rats were anesthetized withketamine (120 mg/kg) and Rompun (10 mg/kg). One of the experimentalgroups received Aranesp 0.1 μg/kg of body weight once on the day beforeinduction of the acute kidney failure. The comparison group comprisedexperimental animals each given an s.c. injection of sodium chloride atthe same time. Blood flow into the kidney was stopped for 45 minutes byplacing an arterial clamp on the right renal arteries. A leftnephrectomy was performed in this time. A sham operation was performedon a further control group. This entailed opening the abdomen, exposingthe left renal artery but not stopping the blood supply, and removingthe contralateral right kidney. All the animals were anesthetized for 60min and sacrificed 24 h after the operation. The 45-minute ischemia withsubsequent reperfusion of the remaining right kidney led to an extensiveacute loss of renal function in the animals treated with sodiumchloride. This is reflected by a serum creatinine level 24 h after theischemia-reperfusion which is 7 times higher than the level before theischemia-reperfusion (p<0.05). By contrast, the animals treated witherythropoietin analog Aranesp showed only a four-fold increase in theserum creatinine levels one day after induction of theischemia-reperfusion damage. There was no increase in the retentionlevels in the animals which underwent left nephrectomy and a shamoperation on the right kidney. The figure shows the creatinineconcentration in the serum of EPO-treated animals (IR+EPO), NaCl-treatedanimals (IR) and sham-operated animals (sham OP) beforeischemia-reperfusion (IR) injury and 24 hours thereafter. It is evidentfrom the figure that the serum creatinine concentration in theAranesp-treated animals is almost halved compared with the controlwithout (NaCl treatment) 24 hours after ischemia-reperfusion injury.

FIG. 7 shows the Kaplan-Mayer survival plots of two experimental groupstreated either with Aranesp or NaCl after induction of chronic renalfailure. 8-week old Sprague Dawley rats were included in the study. Therats were anesthetized with ketamine (120 mg/kg) and Rompun (10 mg/kg).The right kidney was removed from them on day 0 and, immediately afterremoval, was fixed in formalin for histological examination. Thesegmental arteries which supply the upper and lower renal pole of theleft kidney were ligated. This results in a renal infarction of thecorresponding areas of the kidney, and only the middle third of thekidney retains its function. The rats received s.c. injection of Aranesp(0.1 μg/kg of body weight) or NaCl once a week. The animals treated withthe erythropoietin analog Aranesp show a significant survival advantagecompared with the animals treated with sodium chloride (p=0.027; logrank test).

FIGS. 8-15 show optical microscopic kidney sections 6 weeks afterinduction of chronic renal failure in two experimental groups which weretreated either with Aranesp or NaCl and whose Kaplan-Mayer survivalplots are depicted in FIG. 7.

FIG. 8 shows the histological changes in a Sprague-Dawley rat withchronic renal failure after NaCl treatment once a week startingimmediately after induction of the chronic renal failure for a period of6 weeks. The chronic renal failure results from removal of the rightkidney and ligation of the segmental arteries which supply the upper andlower renal pole of the left kidney. The figure shows a medium-sizedpreglomerular artery with characteristic onion ring-type vessel wallproliferation associated with severe hypertensive damage, called Fahr'smalignant nephrosclerosis with endarteritis.

FIG. 9 shows the histological changes in a Sprague-Dawley rat withchronic renal failure after NaCl treatment once a week startingimmediately after induction of the chronic renal failure for a period of6 weeks. The chronic renal failure results from removal of the rightkidney and ligation of the segmental arteries which supply the upper andlower renal pole of the left kidney. The figure shows floridfocal-segmental glomerulosclerosis, called proliferative FSGS (rightglomerulus). The other glomerulus (left) shows ischemic collapse of theloop convolution. A small vessel with severe endothelial damage is to beseen lower in the picture. The observed histological changes correspondto hypertensive organ damage or changes associated with overloadnephropathy following 5/6 nephrectomy.

FIG. 10 shows the histological changes in a Sprague-Dawley rat withchronic renal failure after NaCl treatment once a week startingimmediately after induction of the chronic renal failure for a period of6 weeks. The chronic renal failure results from removal of the rightkidney and ligation of the segmental arteries which supply the upper andlower renal pole of the left kidney. The figure shows almost completesclerosis or destruction of a glomerulus with compensatory enlargementand pronounced hyalinosis or fibrinoid necrosis of the relevant afferentarterioles.

FIG. 11 shows the histological changes in a Sprague-Dawley rat withchronic renal failure after NaCl treatment once a week startingimmediately after induction of the chronic renal failure for a period of6 weeks. The chronic renal failure results from removal of the rightkidney and ligation of the segmental arteries which supply the upper andlower renal pole of the left kidney. The figure shows a smallpreglomerular artery with characteristic onion ring-like vessel wallproliferation and wall necrosis associated with severe hypertensivedamage, called malignant nephrosclerosis (compare right of picture). Anormal (as yet) undamaged arteriole is to be seen on the left.

FIG. 12 shows the histological changes in a Sprague-Dawley rat withchronic renal failure after Aranesp (EPO) treatment (0.1 μg/kg Aranesp)once a week starting immediately after induction of the chronic renalfailure for a period of 6 weeks. The chronic renal failure results fromremoval of the right kidney and ligation of the segmental arteries whichsupply the upper and lower renal pole of the left kidney. The figureshows a normal glomerulus with delicate afferent vessel. There is nopathological tubulointerstitial finding.

FIG. 13 shows the histological changes in a Sprague-Dawley rat withchronic renal failure after Aranesp (EPO) treatment (0.1 μg/kg Aranesp)once a week starting immediately after induction of the chronic renalfailure for a period of 6 weeks. The chronic renal failure results fromremoval of the right kidney and ligation of the segmental arteries whichsupply the upper and lower renal pole of the left kidney. The figureshows a normal glomerulus with delicate afferent vessel (630×magnification). There is no pathological tubulointerstitial finding.

FIG. 14 shows the histological changes in a Sprague-Dawley rat withchronic renal failure after Aranesp (EPO) treatment (0.1 μg/kg Aranesp)once a week starting immediately after induction of the chronic renalfailure for a period of 6 weeks. The chronic renal failure results fromremoval of the right kidney and ligation of the segmental arteries whichsupply the upper and lower renal pole of the left kidney. The figureshows a normal glomerulus with delicate afferent vessel. There is nopathological tubulointerstitial finding.

FIG. 15 shows the histological changes in a Sprague-Dawley rat withchronic renal failure after Aranesp (EPO) treatment (0.1 μg/kg Aranesp)once a week starting immediately after induction of the chronic renalfailure for a period of 6 weeks. The chronic renal failure results fromremoval of the right kidney and ligation of the segmental arteries whichsupply the upper and lower renal pole of the left kidney. The figureshows a normal glomerulus with delicate afferent vessel (630×magnification). There is no pathological tubulointerstitial finding.

EXAMPLE 1 Effect of EPO in Patients with Renal Anemia

The effect of erythropoietin in patients with renal anemia (Hb<10.5g/dl) as a consequence of kidney disease in the terminal stage(preterminal renal failure; creatinine clearance <35 ml/min) wasinvestigated. 11 patients were treated intravenously or subcutaneouslywith erythropoietin in weekly doses averaging 5 000 IU of rhEPO(recombinant human erythropoietin) for a period of at least 8 weeks.After erythropoietin treatment, the endothelial progenitor cells in theblood of the patients were investigated over a period of 20 weeks,analyzing endothelial progenitor cells for their number and theirdifferentiation status by flow cytometry and a culture assay after 0, 2,4, 6 and 8 weeks.

Circulating peripheral blood stem cells (CPBSC) comprise a smallpopulation of cells which express both the CD34 antigen and the CD45antigen. An assay has been developed to determine the number of CPBSC byflow cytometry on the basis of the ISHAGE guidelines (Sutherland et al.,J. Hematother., 5 (1996), 213-226). This assay was used to determineboth the expression pattern of CD34 and CD45 cells and the morphology ofthe stem cells. Both the absolute number of CPBSC per μl and theproportion of CPBSC as a percentage of the total number of leukocyteswas determined in this way.

FIG. 1 shows the results of an FACS analysis of circulating CD34³⁰ stemcells based on the ISHAGE guidelines.

FIG. 2 shows the number of CD34³⁰ stem cells measured by FACS analysisover a period of 8 weeks.

Cell Culture Assay

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficolldensity centrifugation from human blood samples in accordance with themethod described in Asahara, Science, 275 (1997), 964-967. The cellswere plated out on culture plates with fibronectin and maintained in ECbasal medium. EC basal medium consists of EBM-2 basal medium (fromClonetics) and EGM-2 Quots (hEGF; GA-100 (gentamicin, amphotericin-B)FBS, VEGF, hFGF-B (w/heparin), R³-IGF-1, ascorbic acid, heparin). Aftercultivation for 4 days, nonadherent cells were removed by washing theplates. The remaining adherent cells were treated with trypsin andplated out anew. They were then cultivated for a further 3 days. Cellswith the endothelial phenotype were identified by positive staining fortwo different endothelial markers on day 7 after isolation. These areDiI-labeled acetylated low density lipoprotein (acLDL-DiI) and Ulexeuropaeus aglutinin-1 (UEA-1). The results of this investigation aredepicted in FIG. 3.

The results show that erythropoietin is able to mobilize endothelialprogenitor cells and increase the number of circulating endothelialprogenitor cells. Moreover, functional deficits which occur in certainpathological states such as renal anemia are compensated. These resultsare depicted in FIG. 4.

It was found by flow cytometry that the number of circulating CD34³⁰stem cells in patients with renal disease in the terminal stagecorresponds to the number of circulating CD34³⁰ stem cells in the bloodof healthy subjects. After the erythropoietin treatment is started, thenumber of CD34³⁰ stem cells in the bloodstream increases significantlyby more than 50%. It was determined by using the cell culture assay thatthere is a drastic increase in the number of cells developing anendothelial phenotype after treatment with erythropoietin. In afunctional cell culture assay there was an increase of more than 3-foldin the greatly impaired ability of endothelial progenitor cells.

EXAMPLE 2 Improved Wound Healing Through Systemic Use of rhEPO

FVB/N mice were anesthetized by inhalation anesthesia with isoflorane.The fur on the two rear limbs was removed using a depilatory lotion anddisinfected with 70% alcohol. A sterile 4 mm disposable biopsy tissuepunch was used to make a skin wound on the right flank of each of themice. The opposite side served as internal control. Postoperativeantibiotic cover with penicillin G (20 000 units/kg) was performed once.Throughout the period of investigation, subcutaneous injections of therecombinant human erythropoietin analog Aranesp (0.1 μg/kg of bodyweight) took place once a week throughout the study period. Thetreatment started 7 days before removal of the tissue punch. The resultsare depicted in FIG. 5. They show that administration of EPOconsiderably expedites the wound healing process.

EXAMPLE 3 Reduction in the Progression of Chronic Renal Failure ThroughErythropoietin Treatment

8-week-old Sprague-Dawley rats were anesthetized with ketamine (120mg/kg) and Rompun (10 mg/kg). The right kidney was removed from the ratson day 0 and was fixed in formalin immediately after removal forhistological examination. The segmental arteries which supply the upperand lower renal pole of the left kidney were ligated. This results in arenal infarction of the corresponding areas of the kidney, with only themiddle third of the kidney remaining functional. The rats receivedsubcutaneous (s.c.) injection of the erythropoietin analog Aranesp in adose of 0.1 μg/kg of body weight or NaCl as control once a week.

FIG. 7 shows the Kaplan-Mayer survival plots for both experimentalgroups. The Aranesp-treated animals have distinctly improved survivalcompared with the control animals treated with sodium chloride.

FIGS. 12-15 show that the renal tissue shows no pathological changesafter treatment with erythropoietin, whereas severe pathological changesare visible after treatment with NaCl (compare FIGS. 8-11). Furtherhistological investigations revealed that a distinctly greater vesseldensity (CD31) is to be observed in Aranesp-treated animals than inanimals treated with sodium chloride (data not shown).

EXAMPLE 4 Reduction in the Progression of Acute Renal Failure

Sprague-Dawley rats with a body weight of from 250 to 300 g wereemployed for this investigation. One of the experimental groups receivedAranesp in a dose of 0.1 μg/kg of body weight once on the day beforeinduction of the acute kidney failure. The rats were anesthetized withketamine (120 mg/kg of body weight) and Rompun (10 mg/kg). A group ofexperimental animals which received s.c. injection of sodium chloride atthe same time served as comparison. The blood flow in the kidney wasstopped for 45 minutes by placing an arterial clamp on the right renalartery. A left nephrectomy was performed in this time. A sham operationwas performed on a further control group. In this case, the abdomen wasopened, the left renal artery was exposed but the blood supply was notstopped, and the contralateral right kidney was removed. All the animalswere anesthetized for a period of 60 minutes and sacrificed 24 hoursafter the operation.

The 45-minute ischemia with subsequent reperfusion of the remainingright kidney led to extensive acute loss of renal function in theanimals treated with sodium chloride. This was manifested by an increaseby a factor of 7 in the serum creatinine level (p<0.05). By contrast,the animals treated with the erythropoietin analog Aranesp showed only afour-fold increase in the serum creatinine level one day after inductionof the ischemia-reperfusion damage. There was no increase in theretention values in the animals which underwent left nephrectomy and asham operation on the right kidney. The results are depicted in FIG. 6.

1. A method for diabetic wound healing, said method comprisingadministering a pharmaceutical composition comprising a subpolycythemicerythropoietin weekly dose of 1 to 90 international units (IU) EPO/kgbody weight to a subject in need of said wound healing for healing ofsaid diabetic wound.
 2. The method of claim 1, wherein thepharmaceutical composition is administered parenterally.
 3. The methodof claim 2, wherein said parenteral administration is carried out usinga mode selected from the group consisting of intravenous, intramuscular,intracutaneous and subcutaneous, administration.
 4. The method of claim1, where the pharmaceutical composition is administered via pulmonaryadministration.
 5. The method of claim 1, wherein the pharmaceuticalcomposition is orally administered.
 6. The method of claim 1, where thepharmaceutical composition comprises at least one further activeingredient which stimulates endothelial progenitor cells.
 7. The methodof claim 1, wherein the erythropoietin is human or animalerythropoietin.